A vaccine to combat malaria is a highly desirable public health tool to reduce morbidity and mortality in African children. In order to achieve this goal it will be important to gain a detailed understanding of both the nature of the immune response to the current vaccine candidates as well as the immunological status of individuals living in areas in African where malaria is endemic. Over the reporting period this project represented a collaborative effort between Dr. Pierce and Dr. Louis Miller and his colleagues in the Malaria Vaccine Development Unit (MVDU). The immune response at the cellular level was evaluated in individuals in the U.S. enrolled in two clinical trials of the Plasmodium falciparum vaccine candidate, AMA-1 on alum, and one trial of AMA-1 on alum plus the TLR-9 ligand CpG, and in African adults enrolled in parallel studies in Mali. In addition, a study was initiated in Mali to determine the effect of malaria infection on the generation and maintenance of memory B cells. To evaluate the immune status of these individuals, advantage was taken of new and emerging information concerning the function of discrete subsets of lymphocytes in immune responses and the availability of serological reagents to identify these. The B cell antibody response to malaria is believed to be central to the control of parasite infections and thus our initial studies focused on a characterization of B cells in the peripheral blood of individuals enrolled in phase 1 vaccine clinical trials in the U.S. and in Africa. Peripheral blood cells were analyzed by flow cytometry for the B cell markers CD19, CD27, and CD38. Fluorescently labeled AMA-1 was used to identify antigen-specific B cells. In addition, antigen-specific antibody secreting plasma cells were identified by ELISPOT and memory B cells by their response in vitro to the TLR 9 ligand CpG. Cells were analyzed prior to vaccination and at days 3, 7 and 14 following the primary immunization and the secondary immunization (given either 28 or 56 days after the primary). The results acquired thus far showed that the percentage of CD19+ B cells did not change in response to vaccination. However, the number of plasma cells defined as CD27+ and CD38+ and either CD19+ on CD19-, showed an increase between 7 and 14 days after the first vaccination and 3 days after the second vaccination. The effect of vaccination was also mirrored in an increase in the number of memory B cells, defined as CD27+ cells, 3 to 7 days after both the primary and secondary immunization. Preliminary results indicate that the number of antigen-specific memory B cells increased in the periphery after the primary but not after the secondary immunization. Parallel analyses of the B cells in Africans enrolled in a similar Phase I study of AMA-1 or alum showed similar overall B cell response to vaccination. However, memory B cells appeared in the peripheral blood early, at three days post primary vaccination suggesting that individuals living in endemic areas are already immune to AMA-1. Similar analyses are in progress to analyze the memory B cell response to a vaccine containing AMA-1 on alum and CpG in a Phase I trial in the U.S. A similar analyses is planned for both AMA-1 on alum plus CpG Phase I trial in Mali. These results are encouraging indicating that discrete changes in relevant B cell subpopulations can be detected in response to vaccination. These changes may ultimately provide important new parameters to monitor the efficacy of vaccines and guide future vaccine strategies. At present, there is little known about the immunological status at the cellular level of the target population for the vaccine, namely children, in Africa chronically infected with malaria. A detailed analysis of the immune cells in the peripheral blood of chronically infected individuals and a comparison of their profiles with that of nonimmune and vaccinated individuals should provide important new information concerning the repercussion of malaria infection on the immune system and the impact of those effects on the potential to respond to current vaccine candidates. In collaboration with scientists at the MRTC in Mali, we initiated in May 2006, a study to characterize the peripheral blood cells of children and adults in areas in Africa where malaria is endemic. The analyses is a longitudinal study correlating the levels of parasitemia and the immune cell profile in individuals. Lastly, studies will be initiated to determine the effect of malaria infection on both the generation and maintenance of immunological memory. Several anecdotal observations suggest that immunological memory is difficult to establish and only short-lived in individuals living in malaria endemic regions. A clinical protocol will be developed to enroll recent immigrants to the U.S. from West Africa who have malaria infections. These individuals will be treated with anti-malaria drugs and vaccinated for polio and the memory B cell and plasma cells response to polio and to a panel of malaria antigens will be followed with time. The results of this study will hopefully provide new information concerning the generation and maintenance of immunological memory during and following malaria infections.